Assay elements for the rapid analysis of analytes present in biological fluids are known in the art. In one type of such assay elements the sample fluid, e.g., plasma or serum, is applied to the element and the fluid, which contains the analyte of interest, then migrates to a reagent layer or layers. As a result of the interaction between the analyte and the reagent(s) present, a detectable change is brought about in the element which corresponds to the analyte of interest. The detectable change can be a color change which may be evaluated visually or read spectrophotometrically such as with a densitometer. In another scheme based on the presence of fluorescent labelled biological species a fluorescent output signal can be generated and read spectrofluorometrically. In order to obtain accurate and reproducible results with machine readable diagnostic devices it is essential that the plasma or serum be distributed uniformly throughout the assay element so that a uniform signal or color is provided for reading by the instrument.
Various techniques for accomplishing the uniform distribution of the plasma have been suggested in the art. U.S. Pat. No. 3,216,804 discloses an automatic chemical analyzer and sample dispenser and teaches that a uniform sample spot may be obtained by applying a drop of the sample on a filter paper which has fibers extending randomly in all directions or by using porous tapes or membranes. U.S. Pat. No. 3,607,093 discloses a device for testing biological fluids which comprises a liquid permeable membrane of uniform chemical composition which has substantially uniform porosity throughout. U.S. Pat. No. 3,723,064 discloses a multilayer device which has a sample receiving layer having uniform porosity which allows capillary migration to provide an even distribution of the components in the fluid. In cases where an incubation period is required, evaporation from the sample receiving layer could occur with a resultant change in the concentration of analyte in the sample.
U.S. Pat. No. 4,323,536 discloses test devices wherein sample fluid is conveyed to a plurality of test elements by a liquid capillary transport zone. The device comprises a first member, a second covering member, the members having opposing surfaces, and means for spacing the members apart a distance effective to induce capillary flow of liquid introduced between the surfaces and thus create a liquid transport zone. Portions of one or both surfaces may have a plurality of exposed grooves in order to control the liquid flow paths.
Of particular interest among rapid analytical elements are those which are capable of performing the analysis on samples of whole blood since these avoid the need for prior separation of blood cells from plasma such as by centrifuging. In such elements the sample, e.g., a drop of whole blood, is applied to the element which includes some means for separating the cells (erythrocytes, leucocytes) from the plasma and the plasma is then distributed uniformly to a reagent layer or layers so that a uniform signal or color is provided for reading by the instrument.
The known techniques for filtering a whole blood sample and uniformly distributing the plasma have not been entirely satisfactory. In addition to the separation and distribution functions the sample application layer must satisfy a number of other requirements. For example, there must not be any significant amount of binding of the analytes and reagents to the material in the sample application layer, the plasma-analyte concentration level must not be affected, there should be no lysis of the blood cells and the layer must provide a metered amount of plasma to the underlying reagent layers. The known sample application layers and materials fail to provide one or more of these requisite functions.
In an effort to obtain a satisfactory sample application scheme it has been suggested to divide the filtering and distribution functions between different materials. U.S. Pat. No. 4,477,575 discloses a technique for separating cells from plasma or serum which involves applying a sample of whole blood to a layer of glass fibers having an average diameter of 0.2 to 5 microns and a density of 0.1 to 0.5 g/cm.sup.3. There are also disclosed various biological diagnostic devices which incorporate such a glass fiber layer. In one embodiment (see, for example, FIG. 11) the plasma or serum which passes through the filter layer is taken up by a layer of an absorbent material such as cellulose paper or a synthetic fiber fleece which is in contact with the reaction layer. Due to capillary forces the plasma or serum is passed into the reaction layer where the detection reaction takes place.
This arrangement is not satisfactory in all instances. For example, it is not suitable for use with thin film multilayer diagnostic test elements. In such thin film multilayer elements the volume of fluid which is supplied to the test element must be very small and very precisely metered. Since the paper or fiber fleece is relatively thick and has a relatively large surface area the volume of fluid supplied to the test element is relatively large and the precision with which the amount of fluid can be controlled is relatively lower. In addition, because of the area of the relatively thick absorbent material it may give rise to relatively high levels of nonspecific binding of the analyte.
European Patent Application No. 0 160 916 discloses, in an analytical element, a volume filtration layer consisting of a fibrous material and a spreading layer having a liquid retaining capacity which is larger than that of the volume filtration layer. The spreading layer may be a fibrous material, woven cloth, knitted cloth or a non-fibrous porous medium. This arrangement suffers from various of the disadvantages previously discussed. For example, when the spreading layer is a non-fibrous membrane filter the pores of the membrane material are very small and fluid will not pass through easily without the application of pressure.
Accordingly, there is a continuing need for biological diagnostic devices having sample application units which can provide a uniform distribution of plasma or serum to the test element and, in addition, optionally can remove from the sample fluid any components which could interfere with the assay to be performed.